Volume 82, Issue 3, Pages 1632-1643 (March 2002) Transient Exposure of Hydrophobic Surface in the Photoactive Yellow Protein Monitored with Nile Red  Johnny Hendriks, Thomas Gensch, Lene Hviid, Michael A. van der Horst, Klaas J. Hellingwerf, Jasper J. van Thor  Biophysical Journal  Volume 82, Issue 3, Pages 1632-1643 (March 2002) DOI: 10.1016/S0006-3495(02)75514-8 Copyright © 2002 The Biophysical Society Terms and Conditions

Figure 1 Fluorescence emission of Nile Red bound to the pB intermediate of PYP. (A) Emission spectra at pH 5.0, with excitation at 540nm, of NR in the presence of buffer (similar to NR emission in the presence of PYP in the pG state; solid line), and in the presence of a steady-state mixture of pG and pB (0.3/0.7; dotted line). The dashed line was obtainedvia deconvolution and represents the pB-specific NR emission. The residuals after fitting with the multiGauss function (Eq. 1) are shown for the pG (solid line) and the steady-state mixture of pG and pB (dotted line) experiments. (B) NR concentration dependence of the emission spectrum in a PYP sample at pH 5.0 containing a steady-state mixture of pG and pB (0.3/0.7). Spectra are shown for NR concentrations from 100 nM NR (1) to 1μM NR (10). (C) Representative deconvoluted emission spectra at pH 5.0 of NR in buffer (dashed line) and bound to pB (solid line). The dotted line is the deconvoluted pB-associated NR emission spectrum at pH 4.0, which is the only one that deviates significantly of the data obtained in the pH range from 4 to 9. Biophysical Journal 2002 82, 1632-1643DOI: (10.1016/S0006-3495(02)75514-8) Copyright © 2002 The Biophysical Society Terms and Conditions

Figure 2 Quantitative analysis of NR binding to the pB-intermediate of PYP. (A) Determination of the NR binding constant KB at pH 4.0 (▴), pH 5.0 (▾), pH 6.0 (●), pH 7.0 (■), pH 8.0 (◀), and pH 9.0 (▶). Data are plotted so that the slope of each line equals KB (deduced from Eq. 2). (B) pH dependence of the binding constant KB. A proposed fit with the Henderson-Hasselbalch equation using the pKa values of 5.5 (n=3) and 10 (n=0.5) is included. Biophysical Journal 2002 82, 1632-1643DOI: (10.1016/S0006-3495(02)75514-8) Copyright © 2002 The Biophysical Society Terms and Conditions

Figure 3 Steady-state excitation- and emission spectra of NR in a steady-state mixture of pG and pB of wild-type PYP and Δ25-PYP. (A) Fluorescence excitation spectra of NR with detection at 600nm (dashed line) and 659nm (dotted line), selective for pB-associated NR and free NR respectively, in the presence of a steady-state mixture of pG and pB (0.7/0.3) of wild-type PYP at pH 8.0. The emission spectrum (solid line) was recorded with excitation at 540nm. (B) Fluorescence excitation- and emission spectra for a steady-state mixture of pG and pB (0.1/0.9) of Δ25-PYP at pH 8.0. Biophysical Journal 2002 82, 1632-1643DOI: (10.1016/S0006-3495(02)75514-8) Copyright © 2002 The Biophysical Society Terms and Conditions

Figure 4 Comparison of photocycle kinetics determined via NR-emission and UV/Vis absorption. Two types of data are shown, a UV/Vis absorption time trace monitored at 468nm and the amount of NR bound to the pB state of PYP at discrete time points, with a curve fitted through the data points. The data were normalized via the contributions of the recovery exponent obtained in the exponential fit of the data. Both types of data were recorded on the same set-up and with the same amount of PYP and NR present at pH 8.0. Below are the residuals obtained using a monoexponential fit (χ2NR=1.2 · 10−5; χ2abs=9.1 · 10−7; dotted line) and a biexponential fit (χ2NR=6.8 · 10−6; χ2abs=2.0 · 10−7; solid line) of the rise component. Biophysical Journal 2002 82, 1632-1643DOI: (10.1016/S0006-3495(02)75514-8) Copyright © 2002 The Biophysical Society Terms and Conditions

Figure 5 Hydrophobicity analysis of the crystal structures of pG and pB. (A) GRID analysis showing the hydrophobicity of the solvent-accessible molecular surface (calculated with a 1.4-Å probe) of the crystal structure coordinates of the pG state (blue, hydrophobic). (B) GRID analysis showing the hydrophobicity of the solvent-accessible molecular surface of the crystal structure coordinates of the pB state (blue, hydrophobic). (C) Secondary structure representation of PYP, with chromophore and protein (residues 42, 45–52, and 124) coordinates shown for atoms that showed conformational change in the crystalline pB state (blue) relative to the crystalline pG state (yellow). (D) Proposed binding site for Nile Red of PYP in solution in the pB state. The secondary structure representation of residues 42 to 58, 69 to 78, and 95 to 100, that show conformational changes in addition to the N-terminal domain in solution NMR experiments (Craven et al., 2000) are shown in red. The proposed binding site for Nile Red (consisting of some or all of the residues F62, F63, F75, Y76, F79, F92, Y94, F96, V107, and V120) is shown (with carbon atoms in green). The structure coordinate file deposited at the Protein Data Bank (Berman et al., 2000) (http://www.rcsb.org/pdb) with PDB ID: 2PYP (Genick et al., 1997a) was used to prepare the images. All images have the same orientation. Figures were drawn with Aesop (M. Noble, unpublished). Biophysical Journal 2002 82, 1632-1643DOI: (10.1016/S0006-3495(02)75514-8) Copyright © 2002 The Biophysical Society Terms and Conditions